nanoHUB-U Biological Engineering: Cellular Despgn Principles/L2.1: Cellular Architecture: Design Principles of Organelle Size
========================================
 

[Slide 1] Welcome back, I'm Professor Rickus. In this lecture, we're going to talk about cell architecture and the design principles of organelle size. We'll ask what principles and design parameters determine the size cellular of organelles. 

[Slide 2] So specifically, we'll first ponder some questions of organelle size and scaling. For example, the scaling of the volume of the nucleus to volume of the cell. And we'll look in particular at three design principles that control organelle size in cells. Molecular Ruler, looking at the bacteriophage tail as an example. Quantal Synthesis, and Dynamic Assembly, using algae flagella as a model system. 

[Slide 3] So let's first remind ourselves some structural differences between prokaryotes, such as E coli and other bacteria, and eukaryotes, such as the cells in our body. So a typical prokaryotic cell has an outer membrane, it has a cell wall, but no membrane-bound nucleus inside the cell, and lacks many other major organelles. The DNA is in chromosome, as well as in plasmids. So a typical eukaryotic cell has one membrane-bound nucleus. It has two centrioles. It has endoplasmic reticulum organelle. It has multiple mitochondria, many ribosomes. And plants and algae have multiple or many plastids, for example, chloroplasts. 

[Slide 4] So eukaryotic cells can come in many different shapes and sizes, and in a wide range, right? And so one question you can ask is, do bigger cells make more, or do they make bigger organelles? So as some examples of range, here we have one of the smallest known eukaryotic cells, this particular green algae, which is less than one micron in size. On the other end of the spectrum, we have some very large single cells, such as the Xenopus frog egg, which is more than a millimeter in size, right? So again, if you think about the nucleus and think about this question, does a bigger cell have a bigger nucleus? And how do cells sense and control their size and number? And what is the impact of this difference in cell size, from one micron to one millimeter? 

[Slide 5] So these are very fundamental questions of scaling inside cells. Scaling is a very important principle in engineering, as we know. And it is, of course, also an important principle in cells. And this is a fairly old question, and there's been several views looking at this. And here's a quote that I really like that kind of summarizes the problem. So everywhere nature seems to work true to scale, and everything has its proper size accordingly. With these words, D'Arcy Wentworth Thompson elegantly identifies what remains as one of the great mysteries in science, the regulation of the sizes of biological organisms and their substructures. So this field continues today as a very fundamental area of cell biology, biophysics, and biological engineering. 

[Slide 6] So when cells divide, we know that they first double their volume. And cells can divide in different ways. So if we ask question, how does the area scale as the volume scales? And this depends on how the cell divides. So cells can undergo isotropic growth, such as what's approximated in most mammalian cells, right? Us, we're mammals, a type of eukaryotic cell. You can also see long axis growth, such as what would be observed in fission in yeast. And you can also see polarized growth, such as in budding yeast, where this small little bud forms and eventually grows into the size of the original cell. So the area is important, because we know that the surface contains many receptors. These proteins on the cells that are the primary interface with the extracellular environment and the inside signaling of the cell, and ultimately the nucleus and gene expression of the cell. So the surface area to volume ratio may alter intracellular signaling response to environment. And depending on how the cell divides, that area scaling may scale differently than with volume 

[Slide 7] So let's consider transport between the surface and the nucleus of the cell. Let's consider a hypothetical case, if cell two is twice as big as cell one, but has the same nucleus size. So volume scale, so we're looking at sort of isotropic growth here, right, so like our mammalian example. And volume scales with the cubic of the radius, right? So average transport distance between the nucleus and the cell surface increases. So if our nucleus stayed the same size, right? And this average distance between the surface of the nucleus and the surface of the cell would be that traveling distance that would need to be crossed for signals from those membrane receptors to ultimately feed into signalling in the nucleus. And diffusion times scale with the square of distance. So if distance is longer, then passive diffusion times, and therefore signaling times, may increase, okay? And so here we're talking about, if we look a little bit closer here, our case cell 1 has a volume 1. Cell 2 that volume doubles, right? So that radius, right, the radius is going to scale by a factor of 1.26. So radius 2 is 1.26 times radius 1. And that time now, that varies with the square of that distance. So if we look at this long distance here between the surface and the cell is going to scale with the square of that, right? So that time may be 1.6 times longer in our larger cell, where the time is the time for a molecule to traverse that radius of the cell, from the outer to the surface of the nucleus. So, in the cell's surface, as we said, to the nucleus is a major highway of cell signalling. So relaying external information to influence gene expression could be influenced by this volumetric ratio of a cell volume to the nucleus volume. So we can ask the question, is our hypothetical case, is this what we observe in cells? Does the nucleus stay the same? Or does the cell scale the nucleus as the cell volume changes? 

[Slide 8] And before we go on to that, this is just a highlight. We're looking at the nucleus example, but transport is really critical to function of many of the intracellular organelles. And both geometry and scaling will alter their transport. So here I have this table, published from our reference down here, looking at sort of the scaling issue and how different organelles relates to what the transport problem is, such as our nucleus one that we're looking at now. 

[Slide 9] So let's go back and let's ask, as others have done, and look at their work, what happens in real cells? Does the cell scale the nucleus with the cell volume? 

[Slide 10] So this has been looked at in yeast. So here, what these researchers did is that they took yeast under a wide range of conditions. So they have several different mutants here. They put them in different conditions, such as starvation. And under all of these conditions, all these mutants, they saw a wide variety in cell volume. And as you look at cell volume and you look at, then, and measure the volume of the nucleus, they remarkably found that the nucleus to volume ratio is very constant. Even under all these different mutations, all these different conditions such as starvation and other influences that change the volume, the cell is scaling its nucleus volume with its volume. And so you can see that ratio stays fairly constant under all these conditions. And here's some different graphs now plotting the volume of the nucleus versus the volume over the cell. You can see, looking over a wide range of cell volumes, the cell has this linear relation, this slope remains constant. And it's scaling its nucleus with the cell volume. 

[Slide 11] And so these researchers also looked and said, well, what about through the cell cycle? If you remember from previous lecture, we talked about cells changing their mass as they grow. They're dynamic entities, so a cell is not stagnant. It is changing during its growth cycle. And so if they look throughout the cell cycle, right, the volume of the nucleus is increasing as the cell volume is increasing. And again, it is keeping that ratio very constant throughout the cell cycle. 

[Slide 12] So where else might this fundamental question be biologically relevant? What about cancer for example? In cancer cells, a long-noted feature of cancer cells is, the cell nucleus is often enlarged and abnormal in cancer cells. So what would be the biophysical consequence on transport and signalling of an abnormally large nucleus? Or if that ratio that's normally held constant in a cell, if that's abnormally changing throughout the cell? And what are the design principles that are controlling and driving this organelle size, shape, and control? How is the cell measuring its volume or relaying its volume information to control the nucleus volume, or vice versa? 

[Slide 13] So we're going to look at three fundamental design principles that are used in cells to control organelle size. The first one is called the Molecular Ruler. And this is structurally controlled, where a particular protein or structure that has a set size determines the size of an assembling organelle. Second principle is Quantal Synthesis, or is controlled by stoichiometry, where the stoichiometry of starting monomer, that ultimately determines and influences the size of the assembled organelle. And the third principle is Dynamic Assembly, which is kinetically controlled. Where the rates of assembly and disassembly and the equilibrium of those rates results in the control of the ultimate organelle size. 

[Slide 14] So let's first look at principle one, the Molecular Ruler. So again, to reiterate, this is when a length of a particular molecule, usually it's a protein, determines the length of a larger structure. And this is often driven by a self-assembly process, where the organelle is assembling itself. And the molecular ruler tells it when to stop, or determines that ultimate size of when self-assembly terminates. So again, as stated here, the ruler molecule controls when the self-assembly terminates. 

[Slide 15] So the classic model of this is the tail length in bacteriophage. So again, phage are viruses that infect bacteria. Their tail, which is used to inject the genetic material into the host, is made of self-assembled proteins, and they're organized in a disk structure. So lambda phage, for example, has a characteristic tailing. Most phage, each one of those phage particles, has a characteristic length. And the number of discs in that tail determines that ultimate tail length. So the question is, how does the system know when to stop adding disks when the phage is self-assembling inside its bacterial host? When does it know when to stop adding disks to achieve that characteristic tail length? 

[Slide 16] And the first clues from this came years ago from Roger Hendrix, who did his PhD with James Watson, by the way, at Harvard. And he was one of the first to observe that lambda phage with mutations, deletions in a particular gene, Gene H, had shorter tails. 

[Slide 17] So let's look at this work. So they went in and systematic deletions and duplication of sequences, they made these intentionally in Gene H to make them longer or shorter. And the number of disks in the tail they found, then, in these different mutants, of varying Gene H size, correlated linearly with the length of the Gene H product. So they went in and intentionally varied the length of the Gene H product and then measured the tail length, and found this linear correlation. 

[Slide 18] So here's an image of some of the mutants you can see. So here are some of the deletions. You can see the different length of the tail, right? So varying in the number of tail disks from 13 up to wild-type having 32. And they also made an insertion, and saw number of tail disks 39. So it's from this electron microscopy data that you can go back then and correlate the size, the length of the tails with the length of the gene product from Gene H. 

[Slide 19] So how does this work? So we now know that the mechanism of the Gene H product, Gene H acts as a molecular ruler, right, so that protein. And here's how it works. So the tail assembles on an initiator at the capsid head. And this gene product H caps the growing end of the tail and protects from a terminator gene, known as gene product U. So gene product H, the protein from the Gene H, is also attached at the capsid. So you can think of it like a rope attached to the two ends. And as the tail grows, the gene product H, that protein, becomes stretched. And when gene product H is fully extended, the cap pops off the top. And so the end is now no longer protected. And that terminator protein can now come in and cap that end of that tail, and prevent further elongation, further adding of disks, structural proteins to that tail. So the tail, therefore, is fixed at a length that's set by the length of that protein from Gene H. So gene product H, if it gets longer, then more tails or more disks can be added before it pops off. Basically, when it reaches its stretched length, then that determines when it pops off and the terminator comes on. 

[Slide 20] So coming up, we will continue this discussion and go into principles two and principle three, talking about quantal synthesis and dynamic assembly. And we'll specifically look at algae flagella growth as a model system. Hope to see you next time.